The purpose of this project is to elucidate the mechanism of action of the ribosome in protein synthesis by examining the functional roles of individual ribosomal protein and RNA components and the relationships among various partial activities of the ribosome. By means of chemical modification of functional groups, in conjunction with techniques for the in vitro reconstitution of ribosomes from dissociated molecular components, minimal structural alterations will be introduced into the ribosome. The resulting functional lesions will be characterized in detail by means of a variety of partial reaction assays which measure aspects of ribosome activity. The altered molecular component, and in some cases the functional group, responsible for the defect will be identified by constructing ribosomes containing a single modified component. In this way functional roles will be assigned to individual ribosomal proteins and RNA. In addition characterization of a variety of defective ribosomes produced in this way will elucidate the relationships among the partial activities. These experiments will concentrate on the large (50S) ribosomal subunit from Bacillus stearothermophilus. These experiments have identified B-L3 (homologous to Escherichia coli L2) as a possible multifunctional ribosomal protein with functional groups that are essential for several active sites. Experiments are proposed to examine the function of this protein further and determine to what extent its importance for each active site reflects direct participation.